Polymerase chain reaction systems

ABSTRACT

In one embodiment, a polymerase chain reaction (PCR) system includes a mixture chamber, a denature chamber, an annealing chamber, an extension chamber, and a product chamber, that are fluidically coupled to one another through a plurality of microfluidic channels. An inertial pump is associated with each microfluidic channel, and each inertial pump includes a fluid actuator integrated asymmetrically within its associated microfluidic channel. The fluid actuators are capable of selective activation to circulate fluid between the chambers in a controlled cycle.

RELATED APPLICATIONS

This application claims priority from, and incorporates in theirentirety, the following patent applications: Application No.PCT/US2010/035697, filed May 21, 2010; Application Ser. No. 12/833,984,filed Jul. 11, 2010 and issued as U.S. Pat. No. 8,540,355; ApplicationNo. PCT/US2010/043480, filed Jul. 28, 2010; Application No.PCT/US2010/054412, filed Oct. 28, 2010; Application No. PCT/US/054458,filed Oct. 28, 2010; Application No. PCT/US2011/021168, filed Jan. 13,2011; Application No. PCT/US2011/023173, filed Jan. 31, 2011;Application No. PCT/2011/024830, filed Feb. 15, 2011; Application Ser.No. 13/069,630, filed Mar. 23, 2011 and issued as U.S. Pat. No.9,963,739.

BACKGROUND

The polymerase chain reaction (PCR) is a process where a single DNAmolecule can be amplified (replicated) by orders of magnitude intothousands or millions of copies of the molecule. The process relies oncycling a PCR mixture containing polymerase, dNTPs(deoxyribonucleotides), sample DNA template, and primers throughdifferent temperatures. At a first, high-temperature range, denaturationoccurs as the paired strands of the double-stranded sample DNA templateseparate into two individual strands. At a second, low-temperaturerange, annealing of primers complementary to the region of the sampleDNA template being targeted for amplification takes place. At a third,mid-temperature range, extension of the complementary sequence from theprimer occurs, during which the polymerase adheres to the primer anduses nucleotides to replicate each isolated sample DNA template strand.This cycle is typically repeated (e.g., from 20-40 times) to increasethe amount of replicated DNA on an exponential basis until the desiredamount is present for a particular experiment. In general, the PCRamplification process has become an indispensable part of geneticanalysis in various areas including molecular biology, diagnostics,forensics, agriculture, and so on.

Efforts to reduce the time and costs associated with the PCR process areongoing. One area of development is in microfluidic devices whichprovide a miniaturized environment for the PCR process that enables areduction in both the volume of PCR mixture and the time needed for PCRtemperature cycling. Small devices such as microfluidic chips have areduced thermal mass that enables the mixtures to be cycled throughdifferent temperatures in the denaturing, annealing and extending stepsat increased speeds, which reduces the overall time needed forcompleting the PCR process. In addition, increased integration in PCRsystems that include such microfluidic chips has resulted in the use ofmicrofluidic mixers, valves, pumps, channels, chambers, heaters andsensors. However, the pumping and mixing components in such systems aretypically not integrated into the microfluidic chips themselves.Instead, these components are generally external to the microfluidicchip, which increases the size of the integrated system and raises thecosts of fabrication.

BRIEF DESCRIPTION OF THE DRAWINGS

The present embodiments will now be described, by way of example, withreference to the accompanying drawings, in which:

FIG. 1 shows a polymerase chain reaction (PCR) system, according to anembodiment;

FIG. 2 shows a block diagram of an inertial pump-based, PCR architecturesuitable for implementation on a PCR microchip to enable PCR processing,according to an embodiment;

FIG. 3 shows a block diagram of another inertial pump-based, PCRarchitecture suitable for implementation on a PCR microchip to enablePCR processing, according to an embodiment;

FIG. 4 shows an example microchip layout of an inertial pump-based, PCRarchitecture suitable for implementation on a PCR microchip to enablePCR processing, according to an embodiment;

FIG. 5A shows a parallel, linear-type, inertial pump-based, PCRarchitecture suitable for implementation on a PCR microchip, accordingto an embodiment;

FIG. 5B shows a parallel, circular-type, inertial pump-based, PCRarchitecture suitable for implementation on a PCR microchip, accordingto an embodiment;

FIG. 6 shows a parallel, linear-type, inertial pump-based, PCRarchitecture that includes dedicated mixing chambers and is suitable forimplementation on a PCR microchip, according to an embodiment;

FIG. 7 shows another parallel, linear-type, inertial pump-based, PCRarchitecture that includes dedicated mixing chambers suitable forimplementation on a PCR microchip, according to an embodiment;

FIG. 8 shows a parallel, grid-type, inertial pump-based, PCRarchitecture that includes shared mixing chambers and a cleaning system,suitable for implementation on a PCR microchip, according to anembodiment;

FIG. 9 shows an inertial pump integrated in a microfluidic channel thatis suitable for implementing in a PCR architecture of a PCR microchip,according to an embodiment;

FIG. 10 shows a side view of a microfluidic channel with an integratedinertial pump whose fluid actuator is in different stages of operation,according to an embodiment;

FIG. 11 shows the active fluid actuator at the operating stages fromFIG. 10, according to an embodiment;

FIGS. 12, 13 and 14 show the active fluid actuator at the operatingstages from FIG. 10, including net fluid flow direction arrows,according to some embodiments;

FIGS. 15, 16 and 17 show example displacement pulse waveforms, accordingto some embodiments;

FIG. 18 shows a side view of an example microfluidic channel with anintegrated inertial pump whose fluid actuator is in different stages ofoperation, according to an embodiment;

FIG. 19 shows example displacement pulse waveforms whose durationscorrespond with displacement durations of a fluid actuator, according toembodiments; and

FIG. 20 shows an example representation of a fluid actuator deflectingboth into and out of a channel, along with representative displacementpulse waveforms, according to an embodiment.

DETAILED DESCRIPTION Overview of Problem and Solution

As noted above, microfluidic chips are being developed to help reducethe time and costs associated with polymerase chain reaction (PCR). Thetwo basic types of PCR microfluidic chips are a stationary-chamber typechip, and a flow-through type chip. In a stationary-chamber chip, thePCR mixture (including the sample DNA template) is placed in a reactionchamber and temperature cycled without moving the mixture around. Oneproblem with implementing the PCR process in this manner is that the PCRmixture is not the only thing being temperature cycled. Instead, alarger thermal mass that includes the entire mixture, the reactionchamber, and the surrounding environment (i.e., the entire chip) mustall be repeatedly heated and cooled. Disadvantages associated withheating and cooling this large thermal mass include the additional timeand external power needed to complete the temperature cycling.

In a flow-through chip, the PCR mixture is moved through a channel thatrepeatedly passes through the three PCR temperature zones (i.e., in thedenature, annealing, and extension steps). Since the temperature zonescan be heated once and then maintained, only the mixture itself (or evenjust a portion of the mixture) is temperature cycled. The mixturereaches the appropriate temperature in each step of the cycle muchfaster and with much less energy being expended.

However, one disadvantage with the flow-through chip configuration isthat it requires a pump to move the fluidic mixture. Traditional pumpsused in conjunction with flow-through chips have included externalsyringes, pneumatic, peristaltic, rotary, electrophoretic,electromagnetic, electrowetting, and capillary pumps, all of which havevarious disadvantages. For example, external syringes and pneumaticpumps are bulky, non-scalable, cannot be miniaturized, and theyfundamentally limit the complexity of the PCR system, for example, bylimiting the number of external fluidic connections the microfluidicchip can accommodate. Capillary type pumps work on the principle of afluid filling a set of thin capillaries. Therefore, the pump providesonly a single-pass capability. Since the pump is completely passive, theflow of fluid and the number of cycles are “hardwired” into theflow-through chip design and cannot be changed (e.g., throughreprogramming). Electrophoretic pumps can require specialized coatings,complex three-dimensional geometries and high operating voltages, whichlimit the applicability of this type of pump. Other pumps such asperistaltic and rotary pumps have moving parts and are difficult tominiaturize.

In general, therefore, while the use of flow-through microfluidic chipsin PCR systems provides some advantages in reducing costs andtemperature cycling times, they nevertheless suffer disadvantages withregard to the fundamental need to transport fluid. Fluidic movement isfundamental to essential PCR steps such as sample preparation, mixingcomponents, moving the PCR mixture between different temperature zones,disposing of waste, and so on.

Embodiments of the present disclosure improve on prior fluidic pumpsolutions in PCR flow-through microfluidic chips, generally through theuse of micro-inertial pumps integrated within the PCR microchips. PCRmicrochips with integrated micropumps enable programmable and flexiblecycling protocols, fluid flow paths and fluid flow rates in compact PCRmicrosystems. Common microfabrication technologies enable large numbersof such micro-inertial pumps to be fabricated on a single PCR microchip(e.g., in the hundreds or thousands). Numerous microfluidic networkarchitectures are suitable for incorporating micro-inertial pumps inmicrochips that facilitate the PCR process. The integrated micropumpsenable large scale parallel PCR processing on a single PCR microchip.

In one example embodiment, a polymerase chain reaction (PCR) systemincludes a mixture chamber, a denature chamber, an annealing chamber, anextension chamber, and a product chamber, that are fluidically coupledto one another through a plurality of microfluidic channels. An inertialpump is associated with each microfluidic channel, and each inertialpump includes a fluid actuator integrated asymmetrically within itsassociated microfluidic channel. The fluid actuators are capable ofselective activation to circulate fluid between the chambers in acontrolled cycle.

In another example embodiment, a PCR system includes a single premixturechamber to provide PCR solution, a plurality of sample chambers toprovide sample DNA fragments, a temperature cycling area, and aplurality of mixing chambers. Each mixing chamber is fluidically coupledthrough microfluidic channels to the single premixture chamber, thetemperature cycling area, and to a distinct one of the plurality ofsample chambers. The system includes inertial pumps that each have afluid actuator integrated asymmetrically within a microfluidic channelto pump PCR solution and sample DNA fragments into the mixing chambers,and to pump PCR mixture from the mixing chambers through the temperaturecycling area in a controlled cycle. In this embodiment, the system canaccept multiple sample DNA fragments to be mixed with the PCR solutionin dedicated mixing chambers such that the PCR mixture can be amplifiedin the temperature cycling area through the PCR process. Thus, differentDNA samples can be screened for a particular characteristic in parallelor sequentially, since the microfluidic channels/paths of the differentsamples do not overlap.

In another example embodiment, the single premixture chamber in theprevious embodiment is a plurality of premixture chambers and theplurality of sample chambers is a single sample chamber. Each of theplurality of mixing chambers is fluidically coupled through microfluidicchannels to the single sample chamber, the PCR temperature cycling area,and to a distinct one of the premixture chambers. In this embodiment, asingle sample DNA fragment can be screened for multiple characteristics.

In another example embodiment, a polymerase chain reaction (PCR) systemincludes a grid of intersecting microfluidic channels having mixingchambers at each intersection. Premixture chambers are individuallycoupled to microfluidic channels at a first side of the grid, and samplechambers are individually coupled to microfluidic channels at a secondside of the grid. A cleaning agent reservoir is coupled to the gridthrough secondary microfluidic channels that intersect the grid atcleaning junctions. The system also includes inertial pumps having fluidactuators integrated asymmetrically within microfluidic channels to pumpPCR solutions from the premixture chambers into the mixing chambers,sample DNA fragments from the sample chambers into the mixing chambers,PCR mixture from the mixing chambers through a PCR temperature cyclingarea (i.e., denature area, an annealing area, and an extension area),and cleaning solution from the cleaning agent reservoir through the gridof intersecting microfluidic channels.

ILLUSTRATIVE EMBODIMENTS

FIG. 1 shows a polymerase chain reaction (PCR) system, according to anembodiment of the disclosure. The PCR system 100 includes a PCRmicrofluidic chip 102, and may also include external fluid reservoirs104 to supply components of PCR mixtures to the microchip 102. Themicrochip 102 generally includes chambers or areas for introducing,mixing, and temperature cycling the PCR mixtures (polymerase, dNTPs,template DNA sample, and primers). PCR chambers/areas may containtemperature sensors and heating elements, such as resistive heaters.Microchip 102 also includes microfluidic channels formed between thedifferent chambers or areas to fluidically couple them in a manner thatfacilitates the proper temperature cycling sequence in the PCR process.Inertial pumps are integrated within the microfluidic channels ofmicrochip 102 to transport PCR solution (i.e., premixture) and templateDNA to mixing chambers for mixing into PCR mixtures. The inertial pumpsalso transport the PCR mixtures between the different temperaturechambers or areas (i.e., denaturing, annealing, extending) fortemperature cycling, and to product areas when the PCR temperaturecycling is complete. In general, the structures and components of thePCR microchip 102 can be fabricated using conventional integratedcircuit microfabrication techniques such as electroforming, laserablation, anisotropic etching, sputtering, dry etching,photolithography, casting, molding, stamping, machining, spin coatingand laminating.

The PCR system 100 also includes an electronic controller 106 to controlvarious functional aspects of the PCR processing on microchip 102, suchas temperature sensing and control in the reaction chambers/areas, thenumber of temperature cycles a PCR mixture will undergo, and thedirection and rate of flow of the PCR mixture through and between thereaction chambers/areas. Controller 106 typically includes a processor,firmware, software, one or more memory components including volatile andnon-volatile memory components, and other electronics for communicatingwith and controlling components and functions of microchip 102, as wellas controlling fluid reservoir(s) 104. Accordingly, electroniccontroller 106 is a programmable device that typically includesmachine-readable instructions (e.g., PCR process instructions 108) inthe form of one or more software modules, for example, stored in memoryand executable on the controller 106 to control PCR processing onmicrochip 102. Such modules may include, for example, a temperaturesensing and control module 110, a cycle number module 112, a flowdirection module 114, and a rate of flow module 116, as shown in theexample embodiment of FIG. 1.

Electronic controller 106 may also receive data 118 from a host system,such as a computer, and temporarily store the data 118 in a memory.Typically, data 118 is sent to PCR system 100 along an electronic,infrared, optical, or other information transfer path. Data 118represents, for example, executable instructions and/or parameters foruse alone or in conjunction with other executable instructions insoftware/firmware modules stored on electronic controller 106 to controlfluid flow (e.g., PCR mixture flow), temperature cycling, and otherPCR-related functions within microchip 102. For example, varioussoftware and data 118 that are executable on programmable controller 106enable selective and controlled activation of micro-inertial pumps onPCR microchip 102 through precise control over the timing, frequency andduration of compressive and tensile fluid displacements generated byfluid actuators integrated into the microfluidic channels of microchip102. Readily modifiable (i.e., programmable) control over such fluidactuators through data 118 and/or PCR process instructions 108executable on controller 106, allows for any number of different PCRcycling protocols that can be performed on embodiments of PCR microchip102. Such protocols can be readily adjusted on-the-fly for a givenmicrochip 102.

PCR system 100 also typically includes one or more power supplies 120 toprovide power to the PCR microchip 102, electronic controller 106,external fluidic reservoirs 104, and other electrical components thatmay be part of the system 100.

FIG. 2 shows a block diagram of an inertial pump-based, PCR architecture200 suitable for implementation on a PCR microchip 102 to enable PCRprocessing, according to an embodiment of the disclosure. The PCRarchitecture 200 is a functionally if not physically, linearmicrofluidic network that includes a PCR mixture reservoir 202, denaturechamber 204, annealing chamber 206, extension chamber 208, and productoutlet chamber 210, coupled together in a linear fashion by microfluidicchannels. Each microfluidic channel includes, or is part of, an inertialpump configured to move fluidic PCR mixture between chambers in thelinear architecture 200. The inertial pumps in the linear architecture200 can be bidirectional pumps, or, as shown in the FIG. 2 example, theycan be unidirectional pumps such as the unidirectional inertial pumpsbetween the mixture reservoir 202 and denature chamber 204, and betweenthe extension chamber 208 and outlet chamber 210. The pumps in FIG. 2are shown in this manner in order to better illustrate the typical flowof the PCR mixture through the microfluidic channels of the lineararchitecture 200 as discussed below. The operation of bothunidirectional and bidirectional micro-inertial pumps discussedthroughout this disclosure is based on the asymmetric integration(placement) of fluid actuators within the microfluidic channels, as wellas the generation by those fluid actuators of compressive and tensilefluid displacements whose durations are asymmetric (i.e., not equal).The operation of the unidirectional and bidirectional inertial pumps inthis regard is discussed in detail herein below.

In the linear PCR architecture 200, the known PCR process can beimplemented by repeated cycling of PCR mixture through the differenttemperatures of the denature 204, annealing 206 and extension 208chambers. The amount of time and the temperatures illustrated anddiscussed herein regarding the different PCR reaction chambers areintended as examples only. Therefore, while the times and temperaturesare thought to be generally accurate for implementing the known PCRprocess, they may vary. Indeed, as noted above, the describedembodiments contemplate and enable programmable control over these andother factors in a PCR process implemented on a PCR microchip 102.Temperature sensing and temperature control within the different PCRchambers/areas is discussed below with respect to FIG. 4.

In the linear PCR architecture 200, a PCR process can begin when the PCRmixture reservoir 202 receives PCR mixture (polymerase, dNTPs, templateDNA sample, and primers), such as from external reservoir 104, forexample. The PCR mixture reservoir is typically maintained at an ambienttemperature of approximately 25° C. (degrees centigrade). The PCRmixture moves from the PCR mixture reservoir 202 through a microfluidicchannel to the denature chamber 204 by the pumping operation of aunidirectional (or bidirectional) inertial pump. The temperature of thedenature chamber 204 is maintained between approximately 94-96° C., andthe PCR mixture remains in the denature chamber 204 at temperature forapproximately 1 minute. In the denature chamber 204, coiled DNA strands(i.e., of the DNA sample template) separate to form single strands. ThePCR mixture then moves from the denature chamber 204 through amicrofluidic channel to the annealing chamber 206 by the pumpingoperation of a bidirectional inertial pump. The temperature of theannealing chamber 206 is maintained between approximately 55-60° C., andthe PCR mixture remains in the annealing chamber 206 at temperature forapproximately 1 minute. In the annealing chamber 206, primers recognizeor target DNA fragments of interest and bind to both the DNA andcomplementary strands at annealing sites which identify these DNAfragments. This prepares the targeted DNA fragments for DNA synthesis inthe extension phase. The PCR mixture then moves from the annealingchamber 206 through a microfluidic channel to the extension chamber 208by the pumping operation of a bidirectional pump. The temperature of theextension chamber 208 is maintained between approximately 75-80° C., andthe PCR mixture remains in the extension chamber 208 at temperature forapproximately 1 minute. In the extension chamber 208, polymeraseactivates to perform DNA synthesis on the targeted fragments usingnucleotides as building blocks.

When the extension chamber 208 reaction is complete, the PCR mixture hasundergone one PCR temperature cycle, which theoretically has doubled thenumber of targeted DNA fragments. This cycle can then be repeated togenerate an exponential growth in the concentration of the targeted DNAfragment. In the linear PCR architecture 200 of FIG. 2, this cycle isrepeatable by controlling the bidirectional inertial pumps deployedbetween the denature 204, annealing 206 and extension 208 chambers. Morespecifically, a programmable controller 106 is configured to executeinstructions from a flow direction module 114, for example, to controlthe bidirectional pumps such that they move the PCR mixture in anopposite direction from the extension chamber 208 back through theannealing chamber 206 and to the denature chamber 204, where the PCRtemperature cycling process begins again. Additional instructionsexecutable on controller 106 from a cycle number module 112, forexample, direct the controller 106 to cycle the bidirectional pumps aparticular number of times so that the PCR mixture undergoes a desirednumber of temperature cycles. Thus, temperature cycling in the linearPCR architecture 200 of FIG. 2 proceeds in a back-and-forth, linearmanner as the PCR mixture is pumped back and forth between the three PCRreaction chambers (i.e., the denature, annealing and extensionchambers). The number of cycles can be flexibly controlled byprogrammable controller 106 to achieve the desired DNA amplificationresults. When the programmed number of cycles has been completed, thePCR mixture then moves from the extension chamber 208 through amicrofluidic channel to a reaction product outlet chamber 210 by thepumping operation of a unidirectional (or bidirectional) pump. Theproduct outlet chamber 210 is typically maintained at an ambienttemperature such of approximately 25° C.

The PCR temperature cycling process described above with regard to thelinear PCR architecture 200 of FIG. 2 is generally applicable to thevarious embodiments of PCR architectures discussed herein, and willtherefore not be discussed in detail with respect to such otherembodiments.

FIG. 3 shows a block diagram of an inertial pump-based, PCR architecture300 suitable for implementation on a PCR microchip 102 to enable PCRprocessing, according to an embodiment of the disclosure. The PCRarchitecture 300 is a circular microfluidic network in which the threePCR reaction chambers (i.e., the denature 204, annealing 206 andextension 208 chambers) are fluidically coupled in a circular fashion bymicrofluidic channels. Each microfluidic channel includes, or is partof, an inertial pump configured to move fluidic PCR mixture betweenchambers in the circular architecture 300. In this architecture 300, theinertial pumps are all shown as unidirectional pumps. However, theinertial pumps could also be bidirectional pumps. The pumps are shown asunidirectional pumps in order to better illustrate the typical flow ofthe PCR mixture through the microfluidic channels of the circulararchitecture 300. That is, PCR mixture moves (i.e., is pumped) from thePCR mixture reservoir 202 to the denature chamber 204, then to theannealing chamber 206, and then to the extension chamber 208. The PCRmixture is then either pumped back to the denature chamber 204 foradditional temperature cycling, or it is pumped to the product outputchamber 210. Thus, to accomplish typical PCR temperature cyclingprocessing in the circular architecture 300, the use of unidirectionalinertial pumps is sufficient, but not required.

FIG. 4 shows an example microchip layout of an inertial pump-based, PCRarchitecture 400 suitable for implementation on a PCR microchip 102 toenable PCR processing, according to an embodiment of the disclosure. ThePCR architecture 400 is a circular architecture 400 such as that shownin the block diagram of FIG. 3. Accordingly, the PCR architecture 400facilitates the typical flow of PCR mixture as it cycles between PCRreaction chambers through the microfluidic channels, from the PCRmixture reservoir 202 to the denature chamber 204, from the denaturechamber 204 to the annealing chamber 206, from the annealing chamber 206to the extension chamber 208, and then from the extension chamber 208back to the denature chamber 204, where the cycle begins again. Thetemperature cycling continues until a preprogrammed number oftemperature cycles has been completed, after which the PCR mixture movesfrom the extension chamber 208 to the product output chamber 210.

In addition to showing the microfluidic channels and inertial pumps, theexample layout of the PCR architecture 400 also shows resistive heaters402 and temperature sensors 404 in the PCR reaction chambers, as well aselectrodes 406 for energizing the heaters 402, sensors 404 and inertialpumps. In some embodiments, resistive heating elements 402 can alsofunction as temperature sensors when implemented, for example, asthermistors or resistive thermal devices. In such embodiments, separatetemperature sensors would not be needed. As noted above, electroniccontroller 106 controls various functional aspects of the PCR processingon microchip 102, including temperature sensing and control within thereaction chambers/areas. During the temperature cycling process as thecontroller 106 manages the inertial pumps to move PCR mixture betweenthe PCR reaction chambers, it also maintains the temperatures of the PCRreaction chambers (i.e., the denature 204, annealing 206 and extension208 chambers) so that they remain within a specified range thatfacilitates the PCR amplification process. More specifically, theprogrammable controller 106 is configured to execute instructions from atemperature sensing and control module 110, for example, which enablesthe controller 106 to monitor the temperature in each chamber throughtemperature sensors 406. The controller 106 compares the sensedtemperatures to expected temperatures for the different PCR reactionchambers, and if necessary, in response to the comparisons it adjuststhe temperatures to appropriate, programmed, values by selectivelyenergizing resistive heating elements 402.

The example layout of the PCR architecture 400 of FIG. 4 additionallyprovides a general indication about the fabrication of an inertialpump-based, PCR architecture on a microchip 102. As previously noted,the structures and components of the PCR microchip 102 can be fabricatedusing conventional integrated circuit microfabrication techniques suchas electroforming, laser ablation, anisotropic etching, sputtering, dryetching, photolithography, casting, molding, stamping, machining, spincoating and laminating. In general, the resistive elements 402,temperature sensors 404, electrodes 406, and inertial pumps can befabricated on an underlying substrate (e.g., silicon). A channel orchamber layer can then be applied over the substrate, in which themicrofluidic channels and PCR chambers can be formed (i.e., PCR mixturechamber 202, denature chamber 204, annealing chamber 206, extensionchamber 208, and product chamber 210). A top layer can then be appliedover the channel/chamber layer. Both the chamber layer and top layer canbe formed, for example, of a transparent SU8 material commonly used as aphotoresist mask for fabrication of semiconductor devices.

Integrating micro-inertial pumps into microfluidic channels on amicrochip 102 enables the parallelization of the PCR process on amassive scale. FIGS. 5-8 show examples of parallelized, inertialpump-based, PCR architectures suitable for implementation on a PCRmicrochip 102 to enable parallel PCR processing, according to differentembodiments. FIG. 5A shows a parallel, linear-type, inertial pump-based,PCR architecture 500 that is similar to the linear architecturediscussed above with regard to FIG. 2, but which is duplicated N>>1times, according to an embodiment of the disclosure. FIG. 5B shows aparallel, circular-type, inertial pump-based, PCR architecture 502 thatis similar to the circular architecture discussed above with regard toFIG. 3, but which is duplicated N>>1 times, according to an embodimentof the disclosure. It is noted that while the additional parallelembodiments of FIGS. 6-8 show only linear-type, inertial pump-based, PCRarchitectures, corresponding circular-type architectures for theseembodiments are both possible and contemplated by this disclosure,similar to the circular-type architecture explicitly shown in FIG. 5B.

Referring now to FIGS. 5A and 5B, the parallel PCR architectures 500 and502 are suitable for implementation on a microchip 102, and when coupledwith the programmable control of a controller 106 they enable virtuallyunlimited options for PCR processing. For example, in the parallellinear PCR architectures 500 and 502, the PCR mixtures in the N PCRmixture reservoirs 202 can all have different sample DNA templates anddifferent primers, and all the amplification reactions can proceed inparallel. Thus, multiple different DNA samples (i.e., people) can bescreened for a particular gene, one sample can be screened for numerousdifferent genes, several different samples can be screened for numerousdifferent genes, and so on. The PCR processing scenarios enabled bythese parallel architectures 500 and 502 are increased to an evengreater degree by the flexibility in temperature cycling protocols thatcan be readily programmed into and implemented by controller 106. Forexample, controller 106 can implement different cycling protocols withrespect to each of the N linear PCR processes proceeding in parallel inarchitectures 500 and 502 such that each process has a different numberof temperature cycles, a different mixture flow rate, different amountsof time per PCR reaction chamber, and different temperatures within thePCR reaction chambers.

FIG. 6 shows a parallel, linear-type, inertial pump-based, PCRarchitecture 600 that includes dedicated mixing chambers and is suitablefor implementation on a PCR microchip 102, according to an embodiment ofthe disclosure. In this example, N multiple mixing chambers 602 enableindependent mixing of a common PCR premixture solution with differentsample DNA fragments for parallel PCR processing along N linear PCRreaction paths. The parallel PCR architecture 600 includes a single PCRpremixture reservoir 604 in a premixture inlet area to receive a PCRpremixture solution (e.g., from an external reservoir 104) that does notinclude a sample DNA fragment/template. The PCR solution received inpremixture reservoir 604 therefore includes polymerase, dNTPs, andprimers, but no sample DNA fragment. The parallel PCR architecture 600includes N multiple sample DNA inlet reservoirs 606 corresponding withthe N mixing chambers 602, each capable of receiving a sample DNAfragment. Each of the multiple mixing chambers 602 is fluidicallycoupled to a distinct sample reservoir 606 and to the single PCRpremixture reservoir 604 through microfluidic channels.

Microfluidic channels in the parallel PCR architecture 600, as in thepreviously discussed embodiments, include integrated fluid actuatorsthat are asymmetrically located within the channels to form integratedmicro-inertial pumps. The inertial pumps can be bidirectional andsometimes unidirectional, and the integrated fluid actuator of eachinertial pump is individually controllable (e.g., by a controller 106)to generate compressive and tensile fluid displacements havingasymmetric (i.e., unequal) durations that move and circulate fluidbetween different chambers or areas of the parallel PCR architecture600. The control of inertial pumps in the parallel PCR architecture 600can be implemented through instruction modules executable on controller106. For example, controller 106 can execute various instruction modules(e.g., temperature sense and control module 110, cycle number module112, flow direction module 114, flow rate module 116) to implementdifferent PCR temperature cycling protocols with respect to each of theN linear PCR processes proceeding in parallel in architecture 600 suchthat each process has a different number of temperature cycles, adifferent flow rate, different amounts of time per PCR reactionarea/chamber, and different temperatures within the PCR reactionareas/chambers.

In the parallel PCR architecture 600, examples of unidirectionalinertial pumps are those shown between PCR premixture reservoir 604 andmixing chambers 602 that move PCR solution from the PCR premixturereservoir 604 to the chambers 602, and those shown between sample DNAinlet reservoirs 606 and mixing chambers 602 that move fluidic sampleDNA templates from the sample inlet reservoirs 606 to the chambers 602.Examples of bidirectional inertial pumps are those shown between themixing chambers 602 and the PCR reaction area. These bidirectionalinertial pumps move PCR mixtures (i.e., PCR solution after it has beenmixed with a sample DNA fragment/template) from the mixing chambers 602to the PCR reaction area, and then back and forth between the denature,annealing and extension areas (during PCR temperature cycling), and thenon to the reaction product area after the temperature cycling iscomplete. Although not illustrated, the denature, annealing, extensionand product areas may include specific denature, annealing, extensionand product chambers along each of the respective microfluidic channels.In addition, there may also be additional bidirectional inertial pumpsin between each of the denature, annealing and extension areas tofacilitate fluidic movement between these different reaction areas.

In the parallel PCR architecture 600, the sample DNA fragments (DNAtemplates) provided at sample inlet reservoirs 606 can all be different,or they can be common, or they can be some combination thereof. Mixingthe common PCR premixture solution with multiple different samples indedicated mixing chambers 602 of architecture 600 enables, for example,the screening of multiple different patients for the presence of aparticular gene, such as a flu virus. The use of common samples in anumber of different sample inlet reservoirs 606 enables multipleparallel screening of the same sample (i.e., the same patient), whichcan be a useful technique for reducing screening errors.

FIG. 7 shows another parallel, linear-type, inertial pump-based, PCRarchitecture 700 that includes dedicated mixing chambers suitable forimplementation on a PCR microchip 102, according to an embodiment of thedisclosure. In this example, N multiple mixing chambers 702 enableindependent mixing of different PCR premixture solutions with commonsample DNA fragments for parallel PCR processing along N linear PCRreaction paths. In this example, multiple PCR premixture reservoirs 704in a premixture inlet area can receive multiple PCR premixture solutions(e.g., from an external reservoir 104) that do not include a sample DNAfragment, or template. The PCR solutions received in premixturereservoirs 704 therefore includes polymerase, dNTPs, and differentprimers (e.g., primers 1, 2, . . . , N), but no sample DNA fragments.The parallel PCR architecture 700 includes a single sample DNA inletreservoir 706 capable of receiving a sample DNA fragment. Each of themultiple mixing chambers 702 is fluidically coupled to a distinct PCRpremixture reservoir 704 and to the single sample reservoir 706 throughmicrofluidic channels.

Similar to the architectures discussed above (e.g., architectures 200,300, 500, 600), microfluidic channels in the parallel PCR architecture700 include integrated fluid actuators that are asymmetrically locatedwithin the channels to form integrated micro-inertial pumps. Theinertial pumps can be bidirectional and sometimes unidirectional, andthe integrated fluid actuator of each inertial pump is individuallycontrollable (e.g., by a controller 106) to generate compressive andtensile fluid displacements having asymmetric (i.e., unequal) durationsthat move and circulate fluid between different chambers or areas of theparallel PCR architecture 700. The control of inertial pumps in theparallel PCR architecture 700 can be implemented through instructionmodules executable on controller 106. For example, controller 106 canexecute various instruction modules (e.g., temperature sense and controlmodule 110, cycle number module 112, flow direction module 114, flowrate module 116) to implement different PCR temperature cyclingprotocols with respect to each of the N linear PCR processes proceedingin parallel in architecture 700 such that each process has a differentnumber of temperature cycles, a different flow rate, different amountsof time per PCR reaction area/chamber, and different temperatures withinthe PCR reaction areas/chambers.

In the parallel PCR architecture 700, examples of unidirectionalinertial pumps are those shown between PCR premixture reservoirs 704 andmixing chambers 702 that move PCR solutions from the PCR premixturereservoirs 704 to the chambers 702, and those shown between the sampleDNA inlet reservoir 706 and mixing chambers 702 that move a commonfluidic sample DNA fragment/template from the sample inlet reservoir 706to the chambers 702. Examples of bidirectional inertial pumps are thoseshown between the mixing chambers 702 and the PCR reaction area. Thesebidirectional inertial pumps move PCR mixtures (i.e., PCR solutions thathave been mixed with a sample DNA fragment/template) from the mixingchambers 702 to the PCR reaction area, and then back and forth betweenthe denature, annealing and extension areas (during PCR temperaturecycling), and then on to the reaction product area after the temperaturecycling is complete. Although not illustrated, the denature, annealing,extension and product areas may include specific denature, annealing,extension and product chambers along each of the respective microfluidicchannels. In addition, there may also be additional bidirectionalinertial pumps in between each of the denature, annealing and extensionareas to facilitate fluidic movement between these different reactionareas.

In the parallel PCR architecture 700, the PCR solutions from the PCRpremixture reservoirs 704 can all have different primers (e.g., primers1, 2, . . . , N), or the primers can be common, or they can be somecombination thereof. Mixing the different PCR solutions (i.e., withdifferent primers) with common sample DNA fragments in dedicated mixingchambers 702 of architecture 700 enables, for example, screening apatient for the presence of multiple different genes. The use of commonPCR solutions (i.e., with common primers) in PCR in a number of the PCRpremixture reservoirs 704 enables multiple parallel screenings of thecommon sample (i.e., the single patient), which can be useful forreducing screening errors.

FIG. 8 shows a parallel, grid-type, inertial pump-based, PCRarchitecture 800 that includes shared mixing chambers and a cleaningsystem, suitable for implementation on a PCR microchip 102, according toan embodiment of the disclosure. In this example, (M×N) mixing chambers802 (i.e., 802.1.1-802.M.N) serve as intersections in a functional gridthat couples M sample DNA inlet reservoirs 806 with N PCR premixturereservoirs 804 through microfluidic channels. The N PCR premixturereservoirs 804 are fluidically coupled to microfluidic channels at oneside of the grid, while the M sample DNA inlet reservoirs 806 arefluidically coupled to microfluidic channels at another (orthogonal)side of the grid. In the parallel PCR architecture 800, any one of Msamples can be mixed with any of N PCR premixture solutions through amixing chamber 802, and subsequently temperature cycled through the PCRreaction area for amplification. Thus, all M samples can be screened forN different genes or DNA fragments.

In the parallel PCR architecture 800, because the microfluidic channelsand mixing chambers 802 are shared between different samples and PCRsolutions (with different primers), the entire microfluidic network isto be cleaned between successive screenings. Cleaning is implemented bya cleaning system that includes a cleaning agent reservoir 808fluidically coupled to the architecture 800 through a secondary group ofmicrofluidic channels 809 and cleaning junctions 810. As shown in FIG.8, micro-inertial pumps in secondary microfluidic channels 809 betweenthe cleaning agent reservoir 808 and the cleaning junctions 810 pumpcleaning agent from the reservoir 808 through the cleaning junctions810. Inertial pumps on the grid architecture 800 then operate todistribute the cleaning agent further through channels and chambersthroughout the grid architecture 800 to flush out remaining PCR mixturethat may be left over from previous PCR processes. In this manner, amicrochip 102 that includes the parallel architecture 800 can thereforebe used over and over again, and is suitable for integration into a PCRsystem that performs automated PCR processing. Automated processing canproceed, for example, in a sequence beginning with processing a sample1, cleaning the architecture 800, processing a sample 2, cleaning thearchitecture 800, and so on.

Similar to the architectures discussed above (e.g., architectures 200,300, 500, 600, 700), microfluidic channels in the parallel PCRarchitecture 800 include integrated fluid actuators that areasymmetrically located within the channels to form integratedmicro-inertial pumps. The inertial pumps can be bidirectional andsometimes unidirectional as shown in FIG. 8, and the integrated fluidactuator of each inertial pump is individually controllable (e.g., by acontroller 106) to generate compressive and tensile fluid displacementshaving asymmetric (i.e., unequal) durations that move and circulatefluid between different chambers, areas, and junctions of the parallelPCR architecture 800. The control of inertial pumps in the parallel PCRarchitecture 800 is implemented and controlled in a similar manner asdiscussed above with regard to other embodiments. Thus, controller 106executes various instruction modules (e.g., temperature sense andcontrol module 110, cycle number module 112, flow direction module 114,flow rate module 116) to implement different PCR temperature cyclingprotocols. For the PCR architecture 800, one of such PCR temperaturecycling protocols includes a cleaning routine that controls appropriateinertial pumps to circulate cleaning agent from the cleaning agentreservoir 808 through the architecture 800.

Bidirectional and unidirectional inertial pumps in the parallel PCRarchitecture 800 work in the same general manner as noted above withregard to other architectures to move PCR premixtures and samples intomixing chambers 802, and then back and forth between the denature,annealing and extension areas (during PCR temperature cycling), and onto the reaction product area after the temperature cycling is complete.Although not illustrated, the denature, annealing, extension and productareas in architecture 800 may include specific denature, annealing,extension and product chambers along each of the respective microfluidicchannels. In addition, there may also be bidirectional inertial pumps inbetween each of the denature, annealing and extension areas tofacilitate fluidic movement between these different reaction areas.

Inertial Pumps

As noted above, the operation of both unidirectional and bidirectionalmicro-inertial pumps in PCR architectures discussed throughout thisdisclosure is based on the asymmetric integration (placement) of fluidactuators within the microfluidic channels, as well as the generation bythose fluid actuators of compressive and tensile fluid displacementswhose durations are asymmetric (i.e., not equal). Fluid actuatorsintegrated within microfluidic channels at asymmetric locations (i.e.,toward the ends of the channels) can generate both unidirectional andbidirectional fluid flow through the channels. Selective activation ofmultiple fluid actuators located asymmetrically toward the ends ofmultiple microfluidic channels in a network architecture enables thegeneration of directionally-controlled fluid flow patterns within thenetwork. In addition, temporal control over the mechanical operation ormotion of a fluid actuator enables directional control of fluid flowthrough a fluidic network channel. Thus, precise control over theforward and reverse strokes (i.e., compressive and tensile fluiddisplacements) of a single fluid actuator can provide bidirectionalfluid flow within a microfluidic network channel to generatedirectionally-controlled fluid flow patterns within the network.

Fluid actuators can be driven by a variety of actuator mechanisms suchas thermal bubble resistor actuators, piezo membrane actuators,electrostatic (MEMS) membrane actuators, mechanical/impact drivenmembrane actuators, voice coil actuators, magneto-strictive driveactuators, and so on. The fluid actuators and other structures andcomponents of PCR architectures (e.g., PCR reaction chambers,microfluidic channels, etc.) on a PCR microchip 102 can be fabricatedusing conventional integrated circuit microfabrication techniques suchas electroforming, laser ablation, anisotropic etching, sputtering, dryetching, photolithography, casting, molding, stamping, machining, spincoating and laminating.

FIG. 9 shows an inertial pump integrated in a microfluidic channel thatis suitable for implementing in a PCR architecture of a PCR microchip102, according to an embodiment of the disclosure. Referring generallyto FIG. 9, the pumping effect of an inertial pump 900 is based on theaction (i.e., fluid displacements) of a fluid actuator 902 locatedasymmetrically within a fluidic channel 904 (e.g., a microfluidicchannel) whose width is narrower than the width of the reservoir orchamber from, or to, which fluid is being pumped. The asymmetricplacement of the fluid actuator 902 to one side of the center point of afluidic channel 904 establishes a short side 906 of the channel and along side 908 of the channel. Depending on the type of fluid actuatormechanism deployed (see discussion of FIGS. 10-17 below), aunidirectional fluid flow can be achieved in the direction from theshort side 906 (i.e., where the fluid actuator is located) to the longside 908 of the channel. A fluid actuator 902 placed symmetricallywithin a fluidic channel 904 (i.e., at the center of the channel) willgenerate zero or close to zero net flow. Thus, the asymmetric placementof the fluid actuator 902 within the fluidic channel 904 is onecondition that needs to be met in order for an inertial pump 900 toachieve a pumping effect that can generate a net fluid flow through thechannel.

However, in addition to the asymmetric placement of the fluid actuator902 within the fluidic channel 904, another component of the pumpingeffect of an inertial pump 900 is the manner of operation of the fluidactuator 902. Specifically, to achieve the pumping effect and a netfluid flow through the channel 904, the fluid actuator 902 should alsooperate asymmetrically with respect to its displacement of fluid withinthe channel. During operation, a fluid actuator 902 in a fluidic channel904 deflects, first in one direction and then the other (such as the upand down deflections of a flexible membrane or a piston stroke), tocause fluid displacements within the channel. In general, a fluidactuator 902 generates a wave propagating in the fluidic channel 904that pushes fluid in two opposite directions along the channel. If theoperation of the fluid actuator 902 is such that its deflectionsdisplace fluid in both directions with the same speed, then the fluidactuator 902 will generate zero or near zero net fluid flow in thechannel 904. Therefore, in order to generate net fluid flow, theoperation of the fluid actuator 902 should be configured so that itsdeflections, or fluid displacements, are not temporally symmetric. Thatis, an upward deflection into the fluidic channel causing a compressivefluid displacement should not be the same duration as the subsequentdownward deflection causing a tensile fluid displacement. Thus, anasymmetric operation of the fluid actuator with respect to the timing ofits deflection strokes, or fluid displacements, is a second conditionthat needs to be met in order for an inertial pump 900 to achieve apumping effect that can generate a net fluid flow through the channel904.

FIG. 10 shows a side view of a microfluidic channel 1000 with anintegrated inertial pump whose fluid actuator 1002 is in differentstages of operation, according to an embodiment of the disclosure.Fluidic reservoirs or chambers 1004 are connected at each end of thechannel 1000. The integrated fluid actuator 1002 is asymmetricallyplaced at the short side of the channel near an input to a fluidicreservoir 1004, satisfying the first condition needed for an inertialpump to create a pumping effect that can generate a net fluid flowthrough the channel. The second condition that needs to be satisfied tocreate a pump effect is an asymmetric operation of the fluid actuator1002, as noted above. The fluid actuator 1002 is generally describedherein as being a piezoelectric membrane whose up and down deflections(sometimes referred to as piston strokes) within the fluidic channelgenerate fluid displacements that can be specifically controlled (e.g.,by a controller 106). However, a variety of other devices can be used toimplement the fluid actuator including, for example, a resistive heaterto generate a vapor bubble, an electrostatic (MEMS) membrane, amechanical/impact driven membrane, a voice coil, a magneto-strictivedrive, and so on.

At operating stage A shown in FIG. 10, the fluid actuator 1002 is in aresting position and is passive, so there is no net fluid flow throughthe channel 1000, as indicated by the legend. At operating stage B, thefluid actuator 1002 is active and the membrane is deflecting upward intothe fluidic channel 1000. This upward deflection, or forward stroke,causes a compressive (positive) displacement of fluid within the channel1000 as the membrane pushes the fluid outward. At operating stage C, thefluid actuator 1002 is active and the membrane is beginning to deflectdownward to return to its original resting position. This downwarddeflection of the membrane, or reverse stroke, causes a tensile(negative) displacement of fluid within the channel 1000 as it pulls thefluid downward. An upward and downward deflection is one deflectioncycle. A net fluid flow is generated through the channel 1000 if thereis temporal asymmetry between the upward deflection (i.e., thecompressive displacement) and the downward deflection in repeatingdeflection cycles. The question marks in FIG. 10 between opposite netflow direction arrows for the operating stages B and C merely indicatethat the particular temporal asymmetry between the compressive andtensile displacements of the fluid actuator 1002 has not yet beenspecified, and therefore the direction of flow, if any, is not yetknown. Directional flow is discussed below with reference to FIGS.11-14.

FIG. 11 shows the active fluid actuator 1002 at the operating stages Band C from FIG. 10, along with time markers “t1” and “t2” to helpillustrate temporal asymmetry between compressive and tensiledisplacements generated by the fluid actuator 1002, according to anembodiment of the disclosure. The time t1 is the time it takes for thefluid actuator membrane to deflect upward, generating a compressivefluid displacement. The time t2 is the time it takes for the fluidactuator membrane to deflect downward, or back to its original position,generating a tensile fluid displacement. Asymmetric operation of thefluid actuator 1002 occurs if the t1 duration of the compressivedisplacement (upward membrane deflection) is greater or lesser than(i.e., not the same as) the t2 duration of the tensile displacement(downward membrane deflection). Such asymmetric fluid actuator operationover repeating deflection cycles generates a net fluid flow within thechannel 1000. However, if the t1 and t2 compressive and tensiledisplacements are equal, or symmetric, there will be little or no netfluid flow through the channel 1000, regardless of the asymmetricplacement of the fluid actuator 1002 within the channel 1000.

FIGS. 12, 13 and 14 show the active fluid actuator 1002 at the operatingstages B and C from FIG. 10, including net fluid flow direction arrowsthat indicate which direction fluid flows through the channel 1000, ifat all, according to embodiments of the disclosure. The direction of thenet fluid flow depends on the compressive and tensile displacementdurations (t1 and t2) from the actuator. FIGS. 15, 16 and 17 showexample displacement pulse waveforms whose durations correspondrespectively with the displacement durations t1 and t2 of FIGS. 12, 13and 14. For various fluid pump actuators the compressive displacementand tensile displacement times, t1 and t2, can be precisely controlledby a controller 106, for example, executing instructions from aninstruction module 114 (flow direction module 114) within a microfluidicsystem such as a polymerase chain reaction (PCR) system 100 on a PCRmicrochip 102.

Referring to FIG. 12, the compressive displacement duration, t1, is lessthan the tensile displacement duration, t2, so there is a net fluid flowin a direction from the short side of the channel 1000 (i.e., the sidewhere the actuator is located) to the long side of the channel. Thedifference between the compressive and tensile displacement durations,t1 and t2, can be seen in FIG. 15 which shows a corresponding exampledisplacement pulse waveform that might be generated by the fluidactuator with a compressive displacement duration of t1 and a tensiledisplacement duration of t2. The waveform of FIG. 15 indicates adisplacement pulse/cycle on the order of 1 pico-liter (pl) with thecompressive displacement duration, t1, of approximately 0.5 microseconds(ms) and the tensile displacement duration, t2, of approximately 9.5 ms.The values provided for the fluid displacement amount and displacementdurations are only examples and not intended as limitations in anyrespect.

In FIG. 13, the compressive displacement duration, t1, is greater thanthe tensile displacement duration, t2, so there is a net fluid flow inthe direction from the long side of the channel 1000 to the short sideof the channel. The difference between the compressive and tensiledisplacement durations, t1 and t2, can be seen in FIG. 16 which shows acorresponding example displacement pulse waveform that might begenerated by the fluid actuator with a compressive displacement durationof t1 and a tensile displacement duration of t2. The waveform of FIG. 16indicates a displacement pulse/cycle on the order of 1 pico-liter (pl)with the compressive displacement duration, t1, of approximately 9.5microseconds (ms) and the tensile displacement duration, t2, ofapproximately 0.5 ms.

In FIG. 14, the compressive displacement duration, t1, is equal to thetensile displacement duration, t2, so there is little or no net fluidflow through the channel 1000. The equal compressive and tensiledisplacement durations of t1 and t2, can be seen in FIG. 17 which showsa corresponding example displacement pulse waveform that might begenerated by the fluid actuator with a compressive displacement durationof t1 and a tensile displacement duration of t2. The waveform of FIG. 17indicates a displacement pulse/cycle on the order of 1 pico-liter (pl)with the compressive displacement duration, t1, of approximately 5.0microseconds (ms) and the tensile displacement duration, t2, ofapproximately 5.0 ms.

Note that in FIG. 14, although there is asymmetric location of the fluidactuator 1002 within the channel 1000 (satisfying one condition forachieving the pump effect), there is still little or no net fluid flowthrough the channel 1000 because the fluid actuator operation is notasymmetric (the second condition for achieving the pump effect is notsatisfied). Likewise, if the location of the fluid actuator wassymmetric (i.e., located at the center of the channel), and theoperation of the actuator was asymmetric, there would still be little orno net fluid flow through the channel because both of the pump effectconditions would not be satisfied.

From the above examples and discussion of FIGS. 10-17, it is useful tonote the interaction between the pump effect condition of asymmetriclocation of the fluid actuator and the pump effect condition ofasymmetric operation of the fluid actuator. That is, if the asymmetriclocation and the asymmetric operation of the fluid actuator work in thesame direction, the fluid pump actuator will demonstrate a highefficiency pumping effect. However, if the asymmetric location and theasymmetric operation of the fluid actuator work against one another, theasymmetric operation of the fluid actuator reverses the net flow vectorcaused by the asymmetric location of the fluid actuator, and the netflow is from the long side of the channel to the short side of thechannel 1000.

In addition, from the above examples and discussion of FIGS. 10-17, itcan now be better appreciated that the fluid pump actuator 902 discussedabove with respect to the inertial pump 900 of FIG. 9 (shown as aunidirectional inertial pump) is assumed to be an actuator device whosecompressive displacement durations are less than its tensiledisplacement durations. An example of such an actuator is a resistiveheating element that heats the fluid and causes displacement by anexplosion of supercritical vapor. Such an event has an explosiveasymmetry whose expansion phase (i.e., compressive displacement) isfaster than its collapse phase (i.e., tensile displacement). Theasymmetry of this event cannot be controlled in the same manner as theasymmetry of deflection caused by a piezoelectric membrane actuator, forexample. However, as the examples and discussion of FIGS. 10-17 show,the fluid pump actuator 902 of FIG. 9 can also be an actuator devicesuch as a piezoelectric membrane whose fluid displacements can bespecifically controlled by controlling the durations of the up and downdeflections of the membrane within the fluidic channel.

FIG. 18 shows a side view of an example microfluidic channel 1000 withan integrated inertial pump whose fluid actuator 1002 is in differentstages of operation, according to an embodiment of the disclosure. Thisembodiment is similar to that shown and discussed regarding FIG. 10above, except that the deflections of the fluid actuator membrane areshown working differently to create compressive and tensiledisplacements within the channel 1000. At operating stage A shown inFIG. 18, the fluid actuator 1002 is in a resting position and ispassive, so there is no net fluid flow through the channel 1000. Atoperating stage B, the fluid actuator 1002 is active and the membrane isdeflected downward and outside of the fluidic channel 1000. Thisdownward deflection of the membrane causes a tensile displacement offluid within the channel 1000, as it pulls the fluid downward. Atoperating stage C, the fluid actuator 1002 is active and the membrane isbeginning to deflect upward to return to its original resting position.This upward deflection causes a compressive displacement of fluid withinthe channel 1000, as the membrane pushes the fluid upward into thechannel. A net fluid flow is generated through the channel 1000 if thereis temporal asymmetry between the compressive displacement and thetensile displacement. The direction of a net fluid flow is dependentupon the durations of the compressive and tensile displacements, in thesame manner as discussed above.

FIG. 19 shows example displacement pulse waveforms whose durations maycorrespond respectively with displacement durations t1 and t2 of theactuator 1002 of FIG. 18, according to embodiments of the disclosure.The waveforms in FIG. 19 show the tensile (negative) displacementoccurring before the compressive (positive) displacement. In both theprevious examples discussed above, the fluid actuator 1002 begins in aresting position and then either produces a compressive (positive)displacement followed by a tensile (negative) displacement, or itproduces a tensile displacement followed by a compressive displacement.However, various other displacement examples and corresponding waveformsare possible. For example, the fluid actuator 1002 can be pre-loaded ina particular direction and/or it can traverse its resting position suchthat it deflects both into the channel 1000 and out of the channel 1000as it produces compressive and tensile displacements.

FIG. 20 shows an example representation of a fluid actuator 1002deflecting both into and out of a microfluidic channel 1000, along withrepresentative displacement pulse waveforms to illustrate both how theactuator 1002 can deflect into the channel 1000 and out of the channel1000 as it produces compressive and tensile displacements and thepossible pre-loading of the actuator 1002 in a positive or negativedeflection. Such deflections of the actuator 1002 into and out ofchannel 1000 and pre-loading of the actuator 1002 are controlled, forexample, by instruction modules (e.g., flow direction module 114, flowrate module 116) executing on electronic controller 106.

What is claimed is:
 1. A polymerase chain reaction (PCR) systemcomprising: a first chamber and a second chamber fluidically coupled toone another through a microfluidic channel; an inertial pump associatedwith the microfluidic channel associated and including a fluid actuatorintegrated asymmetrically within the microfluidic channel; and acontroller configured to cause circulation of a fluidic PCR mixture fromthe first chamber to the second chamber by repeatedly actuating thefluid actuator over a plurality of deflection cycles between anon-deflected state in which the fluid actuator is not deflected intothe microfluidic channel and a deflected state in which the fluidactuator is deflected into the microfluidic channel, wherein circulationof the fluidic PCR mixture does not occur and the fluid actuator remainsat rest in the non-deflected state when the controller is not actuatingthe fluid actuator.
 2. The PCR system of claim 1, wherein the fluidactuator is thermally driven and electronically controlled.
 3. The PCRsystem of claim 1, wherein the inertial pump comprises a bidirectionalinertial pump and the fluid actuator is controllable to generatecompressive and tensile fluid displacements of varying durations.
 4. ThePCR system of claim 1, wherein the inertial pump is selected from agroup consisting of bidirectional pumps and unidirectional pumps.
 5. ThePCR system of claim 1, wherein actuating the fluid actuators comprisecontrolling compressive and tensile fluid displacement durations of thefluid actuator.
 6. The PCR system of claim 1, further comprising amicrochip on which the chamber, the microfluidic channel and theinertial pump are fabricated.
 7. The PCR system of claim 1, whereinfirst and the second chambers comprise: temperature sensors to sense thetemperatures of the first and the second chambers; and resistive heatersto maintain the temperatures in the first and the second chambers withincontrolled temperature ranges according to sensed temperatures from thetemperature sensors.
 8. The PCR system of claim 7, further comprising: atemperature control module executable on the controller to monitor thetemperature sensors and activate the resistive heaters such that each ofthe first and the second chambers is maintained within a controlledtemperature range.
 9. The PCR system of claim 1, wherein the first andthe second chambers each comprise one of a linear PCR architecture witha denature chamber, an annealing chamber, an extension chamber, and aproduct chamber.
 10. The PCR system of claim 9, comprising a pluralityof the linear PCR architectures on a microchip capable of parallel PCRprocessing.
 11. The PCR system of claim 1, wherein the first and thesecond chambers each comprise one of a denature chamber, a mixturechamber, an annealing chamber, and an extension chamber.
 12. The PCRsystem of claim 11, comprising a plurality of the circular PCRarchitectures on a microchip capable of parallel PCR processing.
 13. Apolymerase chain reaction (PCR) method comprising: repeatedly actuating,over a plurality of deflection cycles, a fluid actuator integratedasymmetrically within a microfluidic channel associated with an inertialpump; circulating a fluidic PCR mixture from a first chamber and asecond chamber fluidically coupled to one another through themicrofluidic channel, via the repeated actuation of the fluid actuatorover the deflection cycles transitioning the fluid actuator between anon-deflected state in which the fluid actuator is not deflected intothe microfluidic channel and a deflected state in which the fluidactuator is deflected into the microfluidic channel, wherein circulationof the fluidic PCR mixture does not occur and the fluid actuator remainsat rest in the non-deflected state when the fluid actuator is not beingactuated.
 14. The PCR method of claim 13, further comprising thermallydriving and electronically controlling the fluid actuator.
 15. The PCRmethod of claim 13, further comprising generating compressive andtensile fluid displacements of varying directions via control of thefluid actuator, the inertial pump being a bidirectional inertial pump.16. The PCR method of claim 13, further controlling activation of thefluid actuator by controlling compressive and tensile fluid displacementdurations of the fluid actuator.
 17. The PCR method of claim 13, furthercomprising: monitoring temperatures of the chambers; activatingresistive heaters associated with the first and second chambers tomaintain the temperatures of the first and second chambers within acontrolled temperature range.
 18. A method comprising: providing a firstchamber and a second chamber; providing a microfluidic channelfluidically coupling the first and second chambers; providing aninertial pump associated with a microfluidic channel and including afluid actuator integrated asymmetrically within the microfluidicchannel; and providing a controller configured to cause circulation of afluidic PCR mixture from the first chamber to the second chamber byrepeatedly actuating the fluid actuator over a plurality of deflectioncycles between a non-deflected state in which the fluid actuator is notdeflected into the microfluidic channel and a deflected state in whichthe fluid actuator is deflected into the microfluidic channel, whereincirculation of the fluidic PCR mixture does not occur and the fluidactuator remains at rest in the non-deflected state when the controlleris not actuating the fluid actuator.
 19. The method of claim 18, whereinthe first and second chambers, the microfluidic channel, and theinertial pump are fabricated on a microchip.